Yeast are important production platforms for the generation of recombinant proteins. In that yeasts are eukaryotes, they share common evolutionary processes with those of higher eukaryotes, including many of the post-translational modifications that occur in the secretory pathway. Recent advances in glycoengineering have resulted in cell lines of the yeast strain Pichia pastoris with genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the process of glycoproteins in humans. See, for example, U.S. Pat. Nos. 7,029,872 and 7,326,681 that describe methods for producing a recombinant glycoprotein in a lower eukaryote host cell that are substantially identical to their human counterparts. Human-like sialylated bi-antennary complex N-linked glycans like those produced in Pichia pastoris from the aforesaid methods have demonstrated utility for the production of therapeutic glycoproteins.
Similar to higher eukaryotes, yeast also express numerous proteases, many of which are either localized to the secretory pathway or pass through it on route to their final destination. As a consequence, non-desirable proteolysis of some recombinant proteins may occur with the specific cleavage being dependent on the class of protease involved. Dipeptidyl aminopeptidases (DAPs) are a class of proteolytic enzymes which remove a two amino acid peptide from the N-terminus of a protein. In Saccharomyces cerevisiae genes for the enzymes STE13 and DAP2 have been identified as having DAP activity, see, Julius et al., Cell, 32: 839-852, 1983; Rendueles et al., J. Bacteriology, 169: 4041-4048, 1987. Applicants herein have developed methods for the elimination of DAP activity in Pichia pastoris, which will allow for the production of full length therapeutic proteins.